Selection, characterization, and biosensing applications of DNA aptamers targeting cyanotoxin BMAA

Scientists have established a connection between environmental exposure to toxins like β-N-methylamino-l-alanine (BMAA) and a heightened risk of neurodegenerative disorders. BMAA is a byproduct from certain strains of cyanobacteria that are present in ecosystems worldwide and is renowned for its bioaccumulation and biomagnification in seafood. The sensitivity, selectivity, and reproducibility of the current analytical techniques are insufficient to support efforts regarding food safety and environment monitoring adequately. This work outlines the in vitro selection of BMAA-specific DNA aptamers via the systematic evolution of ligands through exponential enrichment (SELEX). Screening and characterization of the full-length aptamers was achieved using the SYBR Green (SG) fluorescence displacement assay. Aptamers BMAA_159 and BMAA_165 showed the highest binding affinities, with dissociation constants (Kd) of 2.2 ± 0.1 μM and 0.32 ± 0.02 μM, respectively. After truncation, the binding affinity was confirmed using a BMAA-conjugated fluorescence assay. The Kd values for BMAA_159_min and BMAA_165_min were 6 ± 1 μM and 0.63 ± 0.02 μM, respectively. Alterations in the amino proton region studied using solution nuclear magnetic resonance (NMR) provided further evidence of aptamer–target binding. Additionally, circular dichroism (CD) spectroscopy revealed that BMAA_165_min forms hybrid G-quadruplex (G4) structures. Finally, BMAA_165_min was used in the development of an electrochemical aptamer-based (EAB) sensor that accomplished sensitive and selective detection of BMAA with a limit of detection (LOD) of 1.13 ± 0.02 pM.


Table of Contents
. DNA oligos used for SELEX Table S2.Sequences of the DNA aptamers Table S3.Beads-SELEX scheme for selection of BMAA-specific aptamers Table S4.Identification of overrepresented motifs using AptaTRACE Table S5.Apparent Kd values of the initial screening Figure S3.Screening for binders using the SG fluorescence displacement assay.Table S6.Apparent Kd values were obtained using the SG fluorescence displacement assay.Each measurement represents an independent experiment.The Kd values were determined through non-linear regression analysis by fitting the data with one site-specific binding equation using GraphPad Prism 10.2.0.Table S7.Apparent Kd values were obtained using the BMAA-conjugated fluorescence assay.Each measurement represents an independent experiment.The Kd values were determined through non-linear regression analysis by fitting the data with one site-specific binding equation using GraphPad Prism 10.2.0.

Figure S4 .
Figure S4.Secondary structure of the aptamers with the highest affinity

Figure S3 .
Figure S3.Screening for binders using the SG fluorescence displacement assay.Binding isotherms of A) BMAA_159 (Kd = 3.03) B) BMAA_165 (Kd = 0.234) C) BMAA_172 (Kd = 6.50)D) BMAA_38 (Kd = 18.6).BMAA_96 did not show a concentration-dependent trend within the studied range of concentrations.The Kd values were determined through nonlinear regression analysis by fitting the data with one site-specific binding equation using GraphPad Prism 10.2.0.Control samples of E) BMAA_159 F) BMAA_165 G) BMAA_172 H) BMAA_38.Measurements were performed in triplicates and the error bars represent the calculated standard error.

Figure S4 .Figure S5 .
Figure S4.Secondary structure of the aptamers with the highest affinity A) BMAA 159 B) BMAA_165 C) BMAA_159_min and D) BMAA_165_min.Structures were predicted using the Mfold software considering ambient temperature and ionic conditions of the selection buffer (100 mM NaCl and 2 mM MgCl2).

Figure S6 .
Figure S6.Amino proton region of the 1 H NMR spectra recorded in 20 mM sodium phosphate buffer with 100 mM NaCl and 2 mM MgCl2 at 700 MHz 1 H frequency of A) BMAA_159_min B) BMAA_165_min.Aptamer solutions (20 M) were incubated with increasing molar concentrations of BMAA.Chemical shift perturbations are highlighted, indicating a conformational change in the aptamer upon binding.

Figure S7 .
Figure S7.Independent experiments SG fluorescence displacement assay.Binding isotherms were obtained using GraphPad Prism 10.2.0 for A) BMAA_159 and B) BMAA_165.Measurements were performed in triplicates and the error bars represent the calculated standard error.

Figure S8 .
Figure S8.Independent experiments BMAA-conjugated fluorescence assay.Binding isotherms were obtained using GraphPad Prism 10.2.0 for A) BMAA_159 B) BMAA_165.Measurements were performed in triplicates and the error bars represent the calculated standard error.

Table S1 .
DNA oligos used for SELEX

Table S2 .
Sequences of the DNA aptamers.Overrepresented motifs are underlined.

Table S4 .
Identification of overrepresented motifs using AptaTRACE

Table S5 .
Apparent Kd values of the initial screening were obtained using the SG fluorescence displacement assay.The Kd values were determined through non-linear regression analysis by fitting the data with one site-specific binding equation using GraphPad Prism 10.2.0.No binding refers to the absence of a concentration-dependent trend within the studied range of concentrations.